Probiotics are living microorganisms that are similar to symbiotic microorganisms
found in the human gut. They are also called "friendly bacteria" or "good bacteria"
(Saavedra, 2001; Douglas and Sanders,
2008). According to a definition developed by the Food and Agriculture Organization
of the United Nations (FAO) and World Health Organization (WHO), probiotics
are the microorganisms and, when administered in adequate amounts, confer a
health benefit on the host. (FAO/WHO, 2001). Probiotics
are available to consumers mainly in the form of dietary supplements and foods.
They can be used as Complementary and Alternative Medicine (CAM) (Day,
2002).

Most probiotics are bacteria similar to those naturally found in human guts,
especially in those of breastfed infants who have natural protection against
many diseases (NCCAM, 2008). Most often, the bacteria
come from two groups, Lactobacillus or Bifidobacterium. Within each group, there
are different species such as Lactobacillus acidophilus and Bifidobacterium
bifidus, moreover, within each species, different strains or varieties (Heller,
2001; OMahony et al., 2005).

The concept of probiotics progressed around 1900, when Elie-Metchnikoff hypothesized
that the long and healthy lives of Bulgarian peasants were the outcome of their
consumption of fermented milk and milk products (Kopp-Hoolihan,
2001).

The aim of this study was to investigate the effect of L. acidophilus strains metabolites cultivated in four different types of agro waste substrates namely, pineapple, soy-whey, cabbage and molasses against Staphylococcus aureus (gram positive) and Escherichia coli (gram negative) pathogenic bacteria.

MATERIALS AND METHODS

Microorganisms and culture media reagents: Man-Rogosa-Sharpe (MRS) broth was obtained from (Hi-media, India). Nutrient agar and nutrient broth were purchased from Merck, Darmstadt, Germany. Waste substrates such as pineapple, soy-whey, cabbage and molasses were collected from the local market. The other reagents used in the experiment were analytical grade.

The pathogenic microorganisms, E. coli and S. aureus were obtained from General Hospital. Penang, Malaysia. The bacterial species were cultivated and maintained in nutrient agar slants in screw-capped tubes. The agar slants were preserved in a refrigerator at 4°C until use.

The antimicrobial activities were determined by measuring the diameter of zone of growth inhibition formed around the wells. To further check whether the pathogens were inhibited or killed, a swab was taken from the growth inhibition zone and then inoculated into nutrient broths and incubated at 37°C for 24 h. The broth tubes were then checked for growth. Presence of growth in the broth was interpreted as an inhibitory activity, while no growth was interpreted as microbicidal activity. Each experiment was conducted in two individual trials, each in triplicate.

Statistical analysis: The results were analyzed statistically using one-way analysis of variance (version 13.0, SPSS, USA). When there was a statistically significant difference, post-hoc Tukey Honestly Significant Difference test was applied. ANOVA data with p<0.001, p<0.01 and p<0.05 were classified as statistically significant.

RESULTS AND DISCUSSION

The selection of suitable probiotics starter culture among the one naturally
present in fermented products should be emphasized. Earlier studies showed that
the probiotics grown in modified MRS media exerted considerable antagonistic
effect against the human pathogens Bacillus cereus, Listeria monocytogenes,
MRSA (Methicillin ResistanceStaphylococcus aureus) and Pseudomonas
aeroginosathe causative agents of food borne disease, Listeriosis, skin
infections and lung disease, respectively (Bhilabutra et
al., 2007). Yesillik et al. (2011) showed
that the antibacterial effects of probiotic yoghurt on viability of Salmonella
typhimurium,Escherichia coli, Pseudomonas aeruginosa andStaphylococcus aureuswas investigated by using disc diffusion method. In
their study it was determined that the most sensitive pathogenic bacteria to
the metabolites of probiotics were Salmonella typhimurium where as
the least sensitive pathogen was Pseudomonas aeruginosa.

In the present study, the results of the antibacterial activities of probiotic L. acidophilus strains cultivated in pineapple agro waste juice against S. aureus and E. coli is depicted in Fig. 1. The inhibition zones obtained in S. aureus cultures were in the range of 10.08-10.57 mm, whereas, the inhibition zones observed in E. coli cultures were in the range of 9.77-11.13 mm. The highest inhibition was obtained with L. acidophilus FTDC 4462 against S. aureus (10.57 mm) and E. coli (11.13 mm). While, the lowest inhibition effect was obtained with L. acidophilus FTDC 0582 against S. aureus (10.08 mm) and L. acidophilus FTDC 8592 against E. coli (9.77 mm).

Fig. 1:

The antibacterial activities of probiotic Lactobacillus
acidophilus strains cultivated in pineapple agro waste juice against
Staphylococcus aureus and Escherichia coli. Results are expressed
as Mean±SD; each data point is the average of measurement from six
independent replicates (n = 6). ***: Significant when compared to the control
(p<0.001),**: Significant when compared to the control (p<0.01), *:
Significant when compared to the control (p<0.5)

The antibacterial activities of probiotic Lactobacillus
acidophilus strains cultivated in soy-whey waste against Staphylococcus
aureus and Escherichia coli. Results are expressed as Mean±SD;
each data point is the average of measurement from six independent replicates
(n = 6). Comparison between the different strain grown in the same media
(p<0.05). **: significant when compared to the control (p<0.001),
*: Significant when compared to the control (p<0.01)

Fig. 3:

The antibacterial activities of probiotic Lactobacillus
acidophilus strains cultivated in cabbage waste against Staphylococcus
aureus and Escherichia coli. Results are expressed as Mean±SD;
each data point is the average of measurement from six independent replicates
(n = 6). Comparison between the different strain grown in the same media
(p<0.05). **: Significant when compared to the control (p<0.01), *:
Significant when compared to the control (p<0.5).

Fig. 4:

The antibacterial activities of probiotic Lactobacillus
acidophilus strains cultivated in molases waste against Staphylococcus
aureus and Escherichia coli, Results are expressed as Mean±SD;
each data point is the average of measurement from six independent replicates
(n = 6). Comparison between the different strain grown in the same media
(p<0.05). ***: Significant when compared to the control (p<0.001)

All results were expressed as Mean±SD; each data point is the average of measurement from six independent replicates (n = 6).

The results showed that the metabolites of all eight strains exerted considerable antibacterial effect, as the zone of inhibition results were significant when compared to the control. Among the eight strains cultivated in four different growth medium, there was a statistically significant difference in the anti-bacterial activities of the metabolites of S. aureus and E. coli. Some of the strains of L. acidophilus were more effective against S. aureus, whereas, the other strains were more effective against E. coli.

The results indicated that the agar diffusion method was comparatively more
superior in the study of antimicrobial activity of L. acidophilus strains.
This could be explained by a good diffusion of metabolites from L. acidophilus
strains in the well method and thus this may probably be resulted in the growth
inhibition of the pathogenic microorganisms. The results revealed that the metabolites
from almost all strains tested were microbicidal as there was no growth found
in the cultures collected by swabbing from inhibition zones. It is evident from
the results of the measurement of the diameters of zone of inhibition that the
metabolites are significantly effective. This can be explained from the fact
that the metabolites produced by the probiotics include bioactive products such
as organic acid, hydrogen peroxide (H2O2) and bacteriocins.
It was reported that the cell-free supernatant solution from strains of lactic
acid bacteria exhibited antimicrobial activity which prevented the growth of
different strains of S. aureus and E. coli (Lavermicocca
et al., 2000). It is reported that the principal metabolites of probiotics
bacteria are acetic acid and lactic acid in ratio 3:2 and these acids are responsible
for the consequent drop in pH and may be sufficient to antagonize many pathogenic
bacteria belonging to both Gram-positive and Gram-negative bacteria (Cheikhyoussef
et al., 2007).

The results showed no significant difference in anti-bacterial activities of
L. acidophilus cultivated in pineapple waste substrate, soy-whey substrate
and cabbage waste substrate (p>0.05) whereas, it showed significant differences
with molasses waste substrate (p<0.05). The results of the inhibition zone
diameter of all substrates were considerable and suggested good antimicrobial
activity in these waste substrates against pathogenic bacteria. The major advantages
of these media are that all the tested media are not toxic, in addition they
are suitable for human consumption if they prepared in a suitable formulation
and in contrast, MRS medium was toxic to human. The genus Lactobacillus has
a long history of safe use and it plays a major role in fermented milk and other
food products (Karska-Wysocki et al., 2010).
Huttunen et al. (1995) reported that Lactic acid
bacterial strains are potentially promising because they generate bactericidal
bioactive peptides (bacteriocins) and enzymes that are able to control bio-film
formation and the growth of the pathogens. Certain Lactobacillus strains have
been reported to be highly antagonistic to S. aureus (Ammor
et al., 2006). The current study provides the evidence that the beneficial
effects of probiotics are strain specific. According to the World Gastroenterology
Organisation (WGO) Practice Guidelines on Probiotics and Prebiotics, the
potential probiotic health benefits can only be attributed to the strain or
strains tested and not to the species or the whole group of lactic acid bacteria
or other probiotics (WGO, 2008). Therefore, it is obvious
that L. acidophilus is effective against E. coli and S. aureus
and acted as an bactericidal agent against human pathogenic bacteria. These
results were consistent with the findings of other research groups (Choi
and Beuchat, 1994; Arihara et al., 1996;
Ryan et al., 1996; Aktypis
et al., 1998; Jacobsen et al., 1999;
Parente and Ricciardi, 1999).

CONCLUSION

The growth of the pathogenic bacteria was successfully inhibited when metabolites of L. acidophilus from all waste substrates were tested. There was a significant inhibition of bacterial growth in well diffusion method as depicted by the zone of inhibition. The effect of metabolites was found to be bactericidal on both pathogenic bacteria, E. coli and S. aureus. It can be concluded that L. acidophilus cultivated in waste substrates produced metabolites with strong bactericidal property and could provide medicinally value added advantages to the human beings.

NCCAM, 2008. Get the facts: An introduction to probiotics. National Center for Complementary and Alternative Medicine, U.S. National Institutes of Health. http://www.intute.ac.uk/cgi-bin/fullrecord.pl?handle=20070127-100910.